The long term goal of the proposed research is an understanding of the molecular mechanisms of membrane fusion and targeting in eukaryotic cells and viruses. The major approach is to analyze the fusion reaction of Semliki Forest Virus (SFV), an extremely well-characterized enveloped virus. This virus infects cells via a membrane fusion reaction triggered by the acid pH present in endocytic vesicles. Such a pathway mediates infection by numerous virus families, including those containing such important pathogens as rabies, herpes, and HTLV-III. Endocytosis itself represents a key example of vesicle fusion and targeting, central to the uptake of hormones, nutrients, and growth factors, and the regulation of cell surface receptors. The conformational changes in the fusogenic SFV protein will be analyzed extensively using cloned spike protein genes for wild type virus, and SFV mutants and revertants which have altered pH thresholds. Expression studies, sequence analysis, and genetically engineered chimeras of mutant and wild type spikes will be used to define the role of the individual spike subunits in fusion. SFV fusion is strikingly dependent on the presence of cholesterol in the target membrane. Sterol-depleted cell cultured systems will be used to define the role of sterol in virus infection. Interactions of sterol with the virus spike will be analyzed both biochemically and by generating viral mutants with altered sterol requirements. The fusion activity of SFV will also be used as a tool to follow the origin and movement of endocytic vesicles, using specific depletion or modification of membrane sterol to mark plasma membranes.